This invention relates to materials and methods for detecting and modulating the activity of RNA. The invention generally relates to the field of xe2x80x9cantisensexe2x80x9d compounds which are capable of specific hybridization with a nucleotide sequence of an RNA. In accordance with preferred embodiments, this invention is directed to the design, synthesis, and application of oligonucleotides and to methods for achieving therapeutic treatment of disease, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in a site specific fashion.
It is well known that most of the bodily states in mammals, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to modulate the actual production of such proteins by interactions with the intracellular RNA molecules that code for their synthesis. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides as xe2x80x9cantisensexe2x80x9d agents. The oligonucleotides complementary to a specific target messenger RNA (mRNA) sequence are used. A number of workers have reported such attempts. Pertinent reviews include Stein, et al., Cancer Research 1988, 48, 2659; Walder, Genes and Development 1988, 2, 502; Marcus-Sekura, Anal. Biochemistry 1988, 172, 289; Zon, Journal of Protein Chemistry 1987, 6, 131; Zon, Pharmaceutical Research 1988, 5, 539; Van der Krol, et al., BioTechniques 1988, 6, 958; and Loose-Mitchell, TIPS 1988, 9, 45. Each of the foregoing provide background concerning general antisense theory and prior techniques.
Thus, antisense methodology has been directed to the complementary hybridization of relatively short oligonucleotides to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick base pairs to RNA or single-stranded DNA. The bases of such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides which are designed to bind in a specific fashion toxe2x80x94i.e., which are specifically hybridizable withxe2x80x94a specific mRNA by hybridization. Such analogs are intended to inhibit the activity of the selected mRNAxe2x80x94e.g., to interfere with translation reactions by which proteins coded by the mRNA are producedxe2x80x94by any of a number of mechanisms. It has been hoped to provide therapeutic benefits by inhibiting the formation of the specific proteins which are coded for by the mRNA sequences.
A number of chemical modifications have been introduced into antisense oligonucleotides to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA, and to improve their pharmacokinetic properties. At present, however, no generalized antisense oligonucleotide therapeutic or diagnostic scheme has been found. The most serious deficiency of prior efforts has been the complete lack of a termination event once appropriate hybridization takes place or the occurrence of a termination event that is so inefficient that a useful potency cannot be achieved due to the inability of oligonucleotides to be taken into cells at effective concentrations. The activity of the antisense oligonucleotides presently available has not been sufficient for effective therapeutic, research reagent, or diagnostic use in any practical sense. Accordingly, there has been and continues to be a long-felt need for oligonucleotides which are capable of effective therapeutic and diagnostic antisense use.
This long-felt need has not been satisfied by prior work in the field of antisense oligonucleotide therapy and diagnostics. Others have failed to provide materials which are, at once, therapeutically or diagnostically effective at reasonable concentrations.
Initially, only two mechanisms or terminating events have been thought to operate in the antisense approach to therapeutics. These are the xe2x80x9chybridization arrestxe2x80x9d mechanism (i.e., arrest of translation via antisense hybridization) and the cleavage of hybridized RNA by the cellular enzyme, ribonuclease H (RNase H). It is likely that additional xe2x80x9cnaturalxe2x80x9d events may be involved in the disruption of targeted RNA, however. Other terminating events also have been studied in an attempt to increase the potency of oligonucleotides for use in antisense diagnostics and therapeutics. Thus, an area of research has developed in which a second domain of the oligonucleotide, generally referred to as a pendant group, has been introduced.
The pendant group is not involved with the specific Watson-Crick hybridization of the oligonucleotide with the mRNA but is carried along by the oligonucleotide to serve as a reactive functionality. The pendant group is intended to interact with the mRNA in some manner to more effectively inhibit translation of the mRNA into protein. Such pendant groups have also been attached to molecules targeted to either single or double stranded DNA.
The type of pendant group known as an intercalating agent has been disclosed by Cazenave, et al., Nucleic Acid Research 1987, 15, 4717 and Constant, et al., Biochemistry 1988, 27, 3997. The disclosed purpose of such intercalating agents is to add binding stability to the hybrid formed between the oligonucleotide and the target nucleic acid by binding to the duplex formed between them.
It has also been disclosed to provide a pendant group to oligonucleotides which is capable of cross-linking. Thus, a pendant agent such as psoralen has been disclosed by Yeung, et al., Biochemistry 1988, 27, 2304. It is believed that after hybridization of the oligonucleotide to the target MRNA, the psoralen is photoactivated to cross-link with the mRNA forming a covalent bond between the oligonucleotide and the mRNA, thereby permanently inactivating the MRNA molecule and precluding the further formation of protein encoded by that particular portion of RNA.
It has also been proposed to employ a cross-linking alkylating agent as a pendant group for oligonucleotides for use in antisense approaches to diagnostics and therapeutics, as disclosed by Meyer, J. Am. Chem. Soc. 1989, 111, 8517 and Knorre and Vlassov, Progress in Nucleic Acid Research and Molecular Biology 1985, 32, 291.
The object of employing alkylating agents as pendant groups in oligonucleotides in antisense approaches is to cause the alkylating agent to react irreversibly with the target mRNA. Such irreversible binding between the antisense oligonucleotide and the mRNA is generally covalent and leads to permanent inactivation of the mRNA with a concomitant halt in protein production from the portion of mRNA thus inactivated.
A further strategy which has been proposed is to use chemical reagents which, under selected conditions, can generate a radical species for reaction with the target nucleic acid to cause cleavage or otherwise to inactivate it. Proposed pendant groups of this category include coordination complexes containing a metal ion with associated ligands. A metal ion can change oxidation state to generate reactive oxygen-containing radical ions or other radical species. Doan, et al, Nucleic Acids Research 1987, 15, 8643 have disclosed iron/EDTA and iron/porphyrin species for this purpose. Copper/phenanthroline complexes have been disclosed by Sigman, Accounts of Chemical Research 1986, 19, 180. Dreyer, et al., Proceedings of the National Academy of Sciences, U.S.A. 1985, 82, 968 have investigated the EDTA/Fe moiety to cleave nucleic acids.
Prior approaches using cross-linking agents, alkylating agents, and radical-generating species as pendant groups on oligonucleotides for antisense diagnostics and therapeutics have several significant shortcomings. The sites of attachment of the pendant groups to oligonucleotides play an important, yet imperfectly known, part in the effectiveness of oligonucleotides for therapeutics and diagnostics. Prior workers have described most pendant groups as being attached to a phosphorus atom which, as noted above, affords oligonucleotides with inferior hybridization properties. Prior attempts have been relatively insensitive in that the reactive pendant groups have not been effectively delivered to sites on the messenger RNA molecules for alkylation or cleavage in an effective proportion. Moreover, even if the reactivity of such materials were perfect, i.e. if each reactive functionality were to actually react with a messenger RNA molecule, the effect would be no better than stoichiometric. That is, only one mRNA molecule would be inactivated for each molecule of oligonucleotide. It is also likely that the non-specific interactions of the modified oligonucleotides with molecules other than the target RNA, for example with other molecules that may be alkylated or which may react with radical species, as well as possible self-destruction of the oligonucleotides, not only diminishes the diagnostic or therapeutic effect of the antisense treatment but also leads to undesired toxic reactions in the cell or in vitro. This is especially acute with the radical species which are believed to be able to diffuse beyond the locus of the specific hybridization to cause undesired damage to non-target materials, other cellular molecules, and cellular metabolites. This perceived lack of specificity and stoichiometric limit to the efficacy of such prior alkylating agent and radical generating-types of antisense oligonucleotides is a significant drawback to their employment.
Accordingly, there remains a great need for antisense oligonucleotide formulations which are capable of improved specificity and effectiveness both in binding and in mRNA modulation or inactivation without the imposition of undesirable side effects.
It is one object of this invention to provide oligonucleotides for use in antisense oligonucleotide diagnostics and therapeutics.
It is a further object of this invention to provide such oligonucleotides which are effective in modulating the activity of an RNA.
A further object of this invention is to provide such oligonucleotides which are less likely to evoke undesired or toxic side reactions.
A further object is to provide research and diagnostic methods and materials for assaying bodily states in animals, especially diseased states.
A further object is to provide means for modifying nucleic acids for effecting substitutions on selective portions thereof.
Yet another object is to provide therapeutic and research methods and materials for the treatment of diseases through modulation of the activity of DNA and RNA.
Still another object is to provide means for the selective cleavage of RNA.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and appended claims.
In accordance with the present invention, compositions for modulating the activity of DNA and RNA are provided. The compositions useful for modulating the activity of an RNA or detecting its presence in accordance with this invention generally comprise three portions. The first portion, the targeting portion, is a portion which is specifically hybridizable with a preselected nucleotide sequence of the RNA. The compositions further comprise intercalating portions capable of intercalating between bases or base pairs formed upon hybridization with RNA. The compositions further comprise a reactive portion capable of catalyzing or otherwise effecting the cleavage of RNA, especially of its phosphodiester bonds.
In one embodiment of the invention, preferred compositions according to the present invention comprise at least one ribofuranosyl unit which bears at its 2xe2x80x2 position both an intercalating portion and a reactive portion. The compositions may also include a tether or some other means for connecting the targeting and reactive portions together to form the composition. In a further embodiment of the invention, preferred compositions comprise an oligonucleotide or oligonucleoside including a tether extending from an inter-nucleoside linkage of the oligonucleotide or oligonucleoside. An intercalating portion and a reactive portion portion are connected to the tether. A further tether may be used to connect the intercalating portion to the reactive portion.
The targeting portion of the compositions of this invention preferably comprises oligonucleotides and oligonucleosides including from about 3 to about 50 base units with 8 to 40 subunits being preferred and 12 to 25 being still more preferred. Oligonucleotides and oligonucleosides having about 15 base units are preferable for the practice of certain embodiments of the present invention.
For therapeutic use, preferably, the targeting portion is an analog of an oligonucleotide wherein at least some of the phosphodiester bonds of the oligonucleotide have been substituted with a structure which functions to enhance nuclease resistance and/or to enhance the ability of the compositions to penetrate into the intracellular region of cells where the RNA whose activity is to be modulated is located. In certain preferred embodiments such substitutions comprise phosphorothioate bonds or short chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with other structures having hetero atoms therein forming oligonucleosides.
In certain preferred embodiments, the intercalating portions of the compositions are known, non-carcinogenic types of polycyclic aromatic hydrocarbons or heterocyclic moieties capable of intercalating between predetermined bases and base pairs formed by a hybrid antisense/RNA target sequence duplex.
In accordance with other preferred embodiments the reactive portion of the composition comprises a functionality capable of catalyzing the hydrolysis or cleavage of phosphodiester bonds in RNA. Such functionalities may either be basic, acidic, amphoteric, ionic, or hydrophobic. Heteroatomic species can be formulated to be either basic or acidic or, indeed, to be amphoteric for such purposes. In certain preferred embodiments of the invention, the reactive portion includes multiple ligands capable of coordinating certain metals such as divalent metals including magnesium, calcium and zinc
This invention also comprehends the employment of alkylating and free-radical-forming functionalities as the reactive portions of the subject compositions, particularly where said alkylating or free-radical-forming materials are delivered into the minor groove of the hybrid formed between the compositions of the invention and the RNA to be modulated.
In accordance with other embodiments, the compositions of the invention for modulating the activity of RNA comprise heterocyclic structures having at least one RNA cleaving moiety or some other moiety capable of interacting with an RNA appended thereto. Certain of these compositions are adapted for delivery of the RNA cleaving (i.e., intercalating or minor-groove-binding) moiety to a predetermined portion of the RNA strand, in part by carefully selecting the sites for attachment of the heterocyclic RNA cleaving moieties to the antisense oligonucleotide or analog. Compositions of the invention may include naturally occurring or non-naturally occurring sugar portions, as well as naturally occurring or non-naturally occurring base portions. Accordingly, novel nucleosides and nucleoside analogs are provided. Such nucleosides and nucleoside analogs may be incorporated into oligonucleotides which are useful in the practice of the invention.
The invention also is directed to methods for modulating the activity of an RNA comprising contacting an organism having the RNA with a composition formulated in accordance with the foregoing considerations. It is preferred that the RNA which is to be modulated be preselected to comprise preferably messenger RNA which codes for a protein whose formation is to be modulated. The invention may also be applied to pre-messenger RNA and, indeed, to RNA generically and to single-stranded DNA. The targeting portion of the composition to be employed is selected to be complementary to the preselected portion of RNA or single stranded DNA, that is, to be an antisense oligonucleotide for that portion.
This invention is also directed to methods for treating an organism having a disease characterized by the undesired production or overproduction of a protein, comprising contacting the organism with a composition in accordance with the foregoing considerations, preferably a composition which is designed to specifically bind with messenger RNA which codes for the protein whose production is to be modulated or inhibited.
The invention is also directed to the utilization of groups in addition to the reactive functional groups that are further appended to oligonucleotides. Such pendant groups may lead to enhanced oligonucleotide uptake, enhanced resistance of oligonucleotide to degradation by nucleases, and stronger binding of the oligonucleotides to targeted RNA. Further functionalities may serve to attach reporter groups such as biotin and various fluorophores to sequence-specific oligonucleotides for diagnostic purposes. More than one non-reactive functionality may be attached to each oligonucleotide, two or more non-reactive functionalities may be attached to a single nucleoside unit, and a combination of non-reactive functionalities and reactive functionalities may be attached to a single nucleoside unit or a single oligonucleotide.
Nuclease resistant oligonucleotides of this invention consist of a single strand of nucleic acid bases linked together through linking groups. The target portion of the nuclease resistant oligonucleotide may range in length from about 5 to about 50 nucleic acid bases. However, in accordance with the preferred embodiment of this invention, a target sequence of about 15 bases in length is believed to be optimal.
The bases of the individual nucleotides comprising the oligonucleotides of the invention may be pyrimidines such as thymine, uracil or cytosine, or purines such as guanine or adenine, or both, arranged in a specific sequence. Additionally, they may be any of the synthetic bases known in the art. The sugar moiety of the nucleotides may be of the deoxyribose or ribose type or may be a synthetic sugar known in the art. The phosphate linking groups of the oligonucleotides of the invention may be native or wild type phosphodiester linkages or synthetic linking groups such as, for example, phosphorothioate, phosphorodithioate, methylphosphonate, or alkylphosphonate. Other synthetic linkages that substitute for the phosphate linking groups can also be utilized. For nuclease resistance synthetic linkages are preferred.
The resulting novel oligonucleotides are resistant to nuclease degradation and exhibit hybridization properties of higher quality relative to wild type (DNA-DNA and RNA-DNA) duplexes and the phosphorus modified oligonucleotide antisense duplexes containing phosphorothioates, methylphosphonates, phophoramidates and phosphorotriesters.
The invention further is directed to diagnostic methods for detecting the presence or absence of abnormal RNA molecules or abnormal or inappropriate expression of normal RNA molecules in organisms or cells. It is also directed to methods for the selective cleaving of RNA useful in research and diagnostics. Such selective cleaving is accomplished by interacting RNA with compositions of the invention which have reactive portions capable of effecting such cleavage and targeting portions designed to enforce selectivity.
The invention is also directed to methods for modulating the production of a protein by an organism comprising contacting the organism with a composition formulated in accordance with the foregoing considerations. It is preferred that the RNA or DNA portion which is to be modulated be preselected to comprise that portion of DNA or RNA which codes for the protein whose formation is to be modulated. The targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA, that is, to be an antisense oligonucleotide for that portion.
This invention is also directed to methods of treating an organism having a disease characterized by the undesired production of a protein. This method comprises contacting the organism with a composition in accordance with the foregoing considerations. The composition is preferably one which is designed to specifically bind with messenger RNA which codes for the protein whose production is to be inhibited.
The invention further is directed to diagnostic methods for detecting the presence or absence of abnormal RNA molecules or abnormal or inappropriate expression of normal RNA molecules in organisms or cells.
The invention is also directed to methods for the selective binding of RNA for research and diagnostic purposes. Such selective, strong binding is accomplished by interacting such RNA or DNA with compositions of the invention which are resistant to degradative nucleases and may hybridize more strongly and with greater fidelity than any other known oligonucleotide.
The invention is also directed to in vitro use of certain compounds of the invention as artificial restriction enzymes. Such artificial restriction enzymes are rendered site specific by selection of the appropriate base sequence of the oligonucleotide or oligonucleoside portion of the compound. When so used as in vitro artificial restriction enzymes, the compounds of the inventions can further include a metal ion coordinated to the remainder of the compound via one or more ligand groups selected for specific coordination of a selected metal. Preferred metals are those forming octahedral or tetrahedral coordination complexes. Other metals that can penta-coordinate might also be selected.
In accordance with a further embodiment of the invention, novel processes are provided for the synthesis of novel nucleoside analogs that are substituted in the 2xe2x80x2 position and which are useful for incorporation into oligonucleotides of the invention. Such process provides for introduction of a 2xe2x80x2 substituent in the absence of blocking of either the 3xe2x80x2 or 5xe2x80x2 hydroxyl groups of a ribofuranosyl nucleoside. For adenosine and cytidine, such processes utilize treatment with sodium hydride followed by use of an alkyl halide. For uridine, such processes utilize treatment with stannous chloride and an alkyl halide. For guanosine, such processes treat 2,6-diamino purine riboside with sodium hydride and alkyl halide followed by deamination to the guanosine compound as is disclosed in U.S. patent application Ser. No. 918,362, filed Jul. 23, 1992, the entire disclosure of which is herein incorporated by reference. The reactions are conducted at or near room temperature. These conditions are contrasted to prior known processes that require strong alkylating agents, for instance diazomethane. Such strong alkylating agents necessitate the complete protection of all reactive sites on the nucleoside bases and the 3xe2x80x2 and 5xe2x80x2 sugar hydroxyls.
Certain compositions useful for modulating the activity of an RNA or DNA molecule in accordance with this invention generally comprise a sugar modified oligonucleotide containing a targeting sequence which is specifically hybridizable with a preselected nucleotide sequence of single stranded or double stranded DNA or RNA molecule and which is nuclease resistant.
It is generally desirable to select a sequence of DNA or RNA which is involved in the production of proteins whose synthesis is ultimately to be modulated or inhibited in entirety. The oligonucleotide sequence is synthesized, typically through solid state synthesis of known methodology, to be complementary to or at least to be specifically hybridizable with the preselected nucleotide sequence of the RNA or DNA. Nucleic acid synthesizers are commercially available and their use is generally understood by persons of ordinary skill in the art as being effective in generating nearly any oligonucleotide of reasonable length which may be desired.